In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC II isoforms as well as changes in MHC II isoforms during the course of muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of three nonmuscle MHC II (NMHC II) genes, NMHC II-A, NMHC II-B, and NMHC II-C. We have been studying the transcriptional regulation of NMHC II-A and II-C genes as well as tissue-dependent regulation of alternative splicing of NMHC II-B and C genes. In this report, we focus on regulation of alternative splicing of NMHC II-B and the splicing regulators, Rbfox proteins. The gene encoding NMHC II-B generates alternatively spliced isoforms, which include or exclude a cassette of amino acids (aa) near the ATP-binding domain. Inclusion of alternative exon B1 (also called exon N30) encoding 10 aa in NMHC II-B mRNAs is restricted to some types of neural cells. We have previously reported that two copies of an RNA element UGCAUG located in the intronic region downstream of B1 are required for activation of B1 splicing in neural cells. We also found that Rbfox proteins which contain a conserved RNA recognition motif can bind this RNA element and activate B1 inclusion. There are three genes for Rbfox family proteins in mammals, Rbfox-1 (also called Fox-1 and A2BP1), Rbfox-2 (also called Fox-2, Fxh and Rbm9) and Rbfox-3 (also called Fox-3 and NeuN). Rbfox-1 is expressed in brain and striated muscles whereas Rbfox-2 is expressed in various tissues including brain and muscles. Notably, Rbfox-3 expression is restricted to neural tissues. Comparison of these Rbfox expression patterns with expression of the B1-included NMHC II-B mRNA in the brain and spinal cord demonstrates a better correlation of the extent of B1 inclusion with the level of Rbfox-3 expression rather than with that of Rbfox-1 or 2 expression, although Rbfox-3 and some of the isoforms of Rbfox-1 and 2 are similarly capable of enhancing the B1 splicing when they are over-expressed in cultured cells. To understand a mechanism for Rbfox3-mediated regulation of alternative splicing, we searched for nuclear factor(s) which interact with Rbfox3. We identified the PTB-associated splicing factor (PSF) as an interacting protein with Rbfox3 by affinity-chromatography. In cultured cells, enhancement of B1 inclusion by Rbfox3 depends on the presence of PSF. Rbfox3 is recruited to the UGCAUG element downstream of B1 in the endogenous NMHC II-B transcript in a PSF-dependent manner. Therefore PSF functions as an essential co-activator of Rbfox3. We further extended our research to study the biological function of Rbfox3. To this end, we made use of mouse embryonic carcinoma P19 cells which are capable of differentiating into neuronal cells following retinoic acid treatment. Neuronal differentiation of P19 cells can be monitored by outgrowth of a long axon-like extension which contains an axonal marker, phosphorylated neurofilaments. During neuronal differentiation, expression of Rbfox3 is induced whereas undifferentiated P19 cells do not express Rbfox3. Rbfox1 is barely detected under both undifferentiated and differentiated conditions and the Rbfox2 expression level is unchanged before and after differentiation. The shRNA-mediated knock-down of Rbfox3 results in a decrease in axon-like extensions and an almost complete elimination of phosphorylated neurofilaments. These results indicate that Rbfox3 is required for neuronal differentiation of P19 cells.